Download Molecular methods for detection of probiotics and

Section of Microbiology and Epidemiology
Department of Basic Veterinary Sciences
Faculty of Veterinary Medicine
University of Helsinki
Molecular methods for detection of probiotics and intestinal
microbiota, and evaluation of Lactobacillus brevis as a potential
probiotic dietary adjunct
By Erja Malinen
Academic Dissertation
To be presented, with the permission of the Faculty of Veterinary Medicine of the
University of Helsinki, for public examination in Auditorium Maximum, Hämeentie 57,
00580, Helsinki, on November 29th, 2002 at 12 noon.
Helsinki 2002
Supervised by:
Professor Airi Palva
Section of Microbiology and Epidemiology,
Department of Basic Veterinary Sciences
University of Helsinki
Helsinki, Finland
Reviewed by:
Professor Tapani Alatossava
Department of Food Technology
University of Helsinki
Helsinki, Finland
Associate Professor Finn K. Vogensen
Department of Dairy and Food Science
The Royal Veterinary and Agricultural University
Fredriksberg C, Denmark
ISBN 952-91-5289-2 (paperback)
ISBN 952-10-0783-4 (pdf)
http://ethesis.helsinki.fi
Helsinki 2002
Yliopistopaino
CONTENTS
ABSTRACT .................................................................................................................................................. 4
LIST OF ORIGINAL PUBLICATIONS.................................................................................................... 6
ABBREVIATIONS....................................................................................................................................... 7
1. INTRODUCTION .................................................................................................................................... 8
2. REVIEW OF THE LITERATURE ........................................................................................................ 9
2.1. INTESTINAL MICROBIOTA ..................................................................................................................... 9
2.2. THE PROBIOTICS ................................................................................................................................. 10
2.3. GENETIC LABELLING OF LACTIC ACID BACTERIA ................................................................................ 13
2.4. UTILISATION OF NUCLEIC ACID BASED METHODS FOR IDENTIFICATION AND MONITORING OF BACTERIA
IN POPULATION SAMPLES ........................................................................................................................... 14
2.4.1. Utilisation of methods independent of prior knowledge on sequence data................................. 14
2.4.1.1. Sequencing analysis of 16S ribosomal DNA populations ................................................... 14
2.4.1.2. Denaturing gradient gel electrophoresis (DGGE) and thermal gradient gel electrophoresis
(TGGE)............................................................................................................................................. 15
2.4.1.3. Terminal restriction fragment length polymorphism (T-RFLP) .......................................... 16
2.4.1.4. Analysis of community DNA profiles ................................................................................. 16
2.4.2. Utilisation of specific oligonucleotide primers or probes .......................................................... 17
2.4.2.1. Hybridisation ....................................................................................................................... 17
4.4.2.2. Polymerase chain reaction (PCR)........................................................................................ 18
2.4.2.3. Polymerase chain reaction – enzyme-linked immunosorbent assay (PCR-ELISA) ............ 20
2.4.2.4. Real-time PCR..................................................................................................................... 22
3. AIMS OF THE STUDY ......................................................................................................................... 26
4. MATERIALS AND METHODS ........................................................................................................... 27
4.1. MICROBIAL STRAINS, PLASMIDS, HUMAN CELL LINES AND CULTURE CONDITIONS ............................. 27
4.2. BASIC DNA TECHNIQUES ................................................................................................................... 27
4.3. DESIGN OF OLIGONUCLEOTIDE PRIMERS AND PROBES ........................................................................ 28
4.4. POLYMERASE CHAIN REACTION (I-V) ................................................................................................. 28
4.4.1. PCR-ELISA (II, III)..................................................................................................................... 29
4.4.2. Real-time PCR (IV)..................................................................................................................... 29
4.5. PEPTIDASE ACTIVITY ASSAYS (I) ........................................................................................................ 30
4.6. EVALUATION OF L. BREVIS STRAINS AS DAIRY ADJUNCTS .................................................................. 30
5. RESULTS................................................................................................................................................ 31
5.1. STRAIN-SPECIFIC GENETIC LABELLING OF LACTOBACILLUS HELVETICUS CNRZ32 (I)....................... 31
5.2. SUITABILITY OF PCR-ELISA FOR ANALYSIS OF MIXED DNA POPULATIONS (II, III) .......................... 32
5.2.1. PCR-ELISA with universal primers targeting the 16S and 23S genes (II) ................................. 32
5.2.2. PCR-ELISA with bifidobacterial primers targeting the 16S genes (III) ..................................... 33
5.3. COMPARISON OF REAL-TIME PCR AND DOT BLOT HYBRIDISATION FOR QUANTIFICATION OF THE 16S
RIBOSOMAL DNA OF TARGET BACTERIA (IV) ........................................................................................... 34
5.4. SUITABILITY OF L. BREVIS STRAINS AS DAIRY SUPPLEMENTS (V)....................................................... 35
6. DISCUSSION.......................................................................................................................................... 36
7. CONCLUSIONS..................................................................................................................................... 41
8. ACKNOWLEDGEMENTS ................................................................................................................... 43
9. REFERENCES ....................................................................................................................................... 44
3
Abstract
The human gastrointestinal (GI) tract harbours an extremely complex microbiota
mainly composed of fastidious anaerobic organisms. Because these microbes have a
profound impact on host’s health, modulation of microbiota with probiotic bacteria has
been proposed. However, the mechanisms of action of both the GI microbes and the
proposed probiotics remain obscure. To gain more information, molecular identification
methods for members of the GI microbiota and the probiotic strains are needed. Quick,
robust methods are necessary for obtaining an overall image of changes in GI microbiota,
and more sophisticated methods are needed for following up selected species or strains.
In this study, new methods were tested for their applicability in monitoring GI
microbiota and probiotic strains. Strain-level genetic labelling without introduction of
foreign DNA was demonstrated with Lactobacillus helveticus CNRZ32 by inserting a site
containing silent mutations into the chromosomal pepX gene. Intact phenotypic properties
of the mutated strain were confirmed with a peptidase assay. The mutated and wild-type
strains could be detected from faeces and milk with the help of specific primers. Thus,
the labelling method could be used for specific marking of industrial or probiotic strains
in a ‘food-grade’ manner provided that a suitable target gene and genetic transformation
tools are available.
For analysis of GI microbes and selected intestinal or dairy lactic acid bacteria,
several species- or group-specific oligonucleotide primers and probes were designed and
tested with three different techniques. A polymerase chain reaction – enzyme-linked
immunosorbent assay (PCR-ELISA) application with simultaneous utilisation of multiple
species- or group-specific oligonucleotide probes was suitable for detection of
predominant members present in a mixed bacterial population. Sensitivity of the method
was improved by using primers selective for the genus Bifidobacterium. Comparison of
dot blot hybridisation and real-time PCR demonstrated the superior properties of realtime PCR for detection and quantification of bacterial ribosomal DNA targets.
The final part of this study comprised the evaluation of two Lactobacillus brevis
strains ATCC 8287 and ATCC 14869T as supplements in dairy products. The L. brevis
strains showed promising in vitro antagonistic properties towards selected potentially
harmful microbes and were suitable as supplementary strains in yoghurt, producing no
4
undesirable side effects on the quality or preservation of products. A small-scale feeding
study demonstrated the survival of L. brevis ATCC 8287 in the human GI tract,
indicating, together with the favourable antagonistic properties, that this strain could be a
candidate for use as a probiotic supplement in dairy products.
5
List of original publications
This thesis is based on the following original articles referred to in the text by their
Roman numerals.
I
Malinen, E., Laitinen, R., Palva, A. (2001). Genetic labeling of lactobacilli in
food grade manner for strain-specific detection of industrial starters and probiotic
strains. Food Microbiology 18, 309-317.
II
Laitinen, R., Malinen, E., Palva, A. (2002). PCR-ELISA I: Application to
simultaneous analysis of mixed bacterial samples composed of intestinal species.
Systematic and Applied Microbiology 25, 241-248.
III
Malinen, E., Mättö, J., Salmitie, M., Alander, M., Saarela, M., Palva, A. (2002).
PCR-ELISA II: Analysis of Bifidobacterium populations in human faecal samples
from a consumption trial with Bifidobacterium lactis Bb-12 and a galactooligosaccharide preparation. Systematic and Applied Microbiology 25, 249-258.
IV
Malinen, E., Kassinen, A., Rinttilä, T., Palva, A. (2002). Comparison of real-time
PCR with SYBR Green I or 5´-nuclease assays and dot-blot hybridization with
rDNA targeted oligonucleotide probes in quantification of selected faecal
bacteria. Microbiology (in press).
V
Rönkä, E., Malinen, E., Saarela, M., Rinta-Koski, M., Aarnikunnas, J., Palva, A.
(2002). Probiotic and milk technological properties of Lactobacillus brevis.
International Journal of Food Microbiology (in press).
6
Abbreviations
ATCC
American Type Culture Collection
DGGE
denaturing gradient gel electrophoresis
DNA
deoxyribonucleic acid
EBI
European Bioinformatics Institute
FISH
fluorescence in situ hybridisation
G+C
guanine-plus-cytosine
GI
gastrointestinal
GRAS
generally recognized as safe
IL
interleukin
PCR
polymerase chain reaction
PCR-ELISA
polymerase chain reaction – enzyme–linked
immunosorbent assay
RNA
ribonucleic acid
rDNA
ribosomal DNA
rRNA
ribosomal RNA
SSCP
single-strand conformation polymorphism
TGGE
thermal gradient gel electrophoresis
TRF
terminal restriction fragment
T-RFLP
terminal restriction fragment length polymorphism
7
1. Introduction
Molecular
methods
have
facilitated
culture-independent
studies
of
gastrointestinal (GI) tract microbes. The GI microbiota is mainly composed of anaerobic
organisms (Rolfe, 1997) and moreover, direct molecular approaches have confirmed the
abundance of uncultivable microbes in intestinal samples (Langendijk et al., 1995; Suau
et al., 1999; Leser et al., 2002). The value of molecular methods for studying GI tract
microbes is therefore immense.
GI microbiota can be influenced by probiotic bacteria (Fuller, 1989; Dunne et al.,
1999). The probiotics, defined as “live microbial food supplements which beneficially
affect the host by improving the intestinal microbial balance” (Fuller, 1989), have been
proposed to possess several advantageous properties, such as antagonistic actions,
production of antimicrobial substances, modulation of immune responses and an impact
on the metabolic activities of the gut (Dunne et al., 1999). Probiotic bacteria seem to hold
great promise for treatment of gastrointestinal disorders, yet further studies are required
to create a more scientific basis for the probiotic action. Furthermore, as no single strain
is likely to have all the aforementioned beneficial properties (Dunne et al., 1999),
screening of new strains for probiotic potential is necessary.
Although several applications, such as dot blot hybridisation (Doré et al., 1998),
fluorescence in situ hybridisation (FISH) (Langendijk et al., 1995), denaturing gradient
gel electrophoresis (DGGE) (Millar et al., 1996) or thermal gradient gel electrophoresis
(TGGE) (Zoetendal et al., 1998), and polymerase chain reaction (PCR) with species- or
group-specific primers (Kok et al., 1996), are available for the detection and semiquantitative analysis of GI microbiota, improvements are required, especially in relation
to sensitivity, cost and quantification power of the methods. This is fundamental for
better understanding of the GI tract microbiota and the effects of probiotic bacteria on
these microbes.
8
2. Review of the literature
2.1. Intestinal microbiota
The human gastrointestinal (GI) tract harbours an extremely complex microbiota
which has a profound impact on host’s health. The normal gut bacterial population of an
adult is estimated to comprise more than 400 species, with a predominance of obligate
anaerobes (Rolfe, 1997). The total number of microbes present in one gram of intestinal
content varies from less than 103 microbes in the stomach to 104 – 107 microbes in the
small intestine and 1010 – 1012 microbes in the colon (Goldin and Salminen, 1998;
Holzapfel et al., 1998). Indeed, the quantity of microbes present in the intestine (about
1014) exceeds 10-fold the total number of all human cells (Holzapfel et al., 1998). Until
recently, analysis of intestinal bacteria has mainly been based on cultivation-dependent
methods. Culture-independent studies have however confirmed that only a fraction of the
organisms present in faeces are cultivable (Langendijk et al., 1995; Suau et al., 1999;
Leser et al., 2002), therefore, the results obtained by cultivation are likely to be biased.
Generally, Bacteroides,
Eubacterium, Clostridium, Ruminococcus,
Peptococcus,
Peptostreptococcus, Bifidobacterium and Fusobacterium are reported to constitute the
majority of microbiota. Molecular analyses, however, suggest that most faecal bacteria
belong to a few phylogenetic lineages composed of organisms from several genera (Suau
et al., 1999, Sghir et al., 2000). Bacteria related to Bacteroides, Prevotella and
Porphyromonas seem to represent one-third of bacteria present in faeces, whereas
Clostridium leptum subgroup and Clostridium coccoides groups both account for
approximately one-fifth of the faecal bacterial populations (Sghir et al., 2000). However,
besides being an extremely diverse microbial ecosystem, the intestinal microbiota
appears to be unique for each individual (Kimura et al., 1997; Tannock, 1999a).
The normal microbiota of the GI tract works as a barrier against pathogens,
contributes to degradation of some food components, stimulates the host immune system,
and produces certain B vitamins, enzymes and short-chain fatty acids (Holzapfel et al.,
1998). The microbes can also metabolise potentially carcinogenic substances and drugs in
either a beneficial or a disadvantageous way (Holzapfel et al., 1998). However, the role
and action of individual microbial species or groups present in the GI tract are poorly
9
known. Starting at birth, the microbiota develops in a successional manner. The first
colonising microbes include bifidobacteria, enterobacteria (Grönlund et al., 2000; Favier
et al., 2002), clostridia, enterococci and ruminococci (Favier et al., 2002). Recent studies
have suggested the importance of the type of colonising bacteria on the development of
the gut immune system (Grönlund et al., 2000; Ouwehand et al., 2001a). A role in the
development of food allergies has been postulated for the intestinal microbiota due to the
occurrence of an adult-like bifidobacterial species composition in the intestine of allergic
infants (Ouwehand et al., 2001a). Significant structural changes occur in the microbiota
with age, including reduction of bifidobacteria (Hopkins et al., 2001) as well as an
increase in the diversity of Atopobium cluster species (Harmsen et al., 2000b) present in
faeces. Nutritional aspects, however, have a profound effect on the composition of the
intestinal microbial population (Benno et al., 1989).
2.2. The Probiotics
Probiotic bacteria have been defined as “live microbial food supplements which
beneficially affect the host by improving the intestinal microbial balance” (Fuller, 1989).
Probiotic bacteria are increasingly utilised in human food as well as in animal feed
products (Fuller, 1999; Sanders and Huis in’t Veld, 1999). However, composition of the
intestinal microbiota is poorly known, which hinders understanding of the probiotic
functions (Tannock, 1999b). A probiotic strain should be of host origin, non-pathogenic,
technologically suitable for industrial processes, acid- and bile-fast, adhere to the gut
epithelial tissue, persist in the gastrointestinal tract for short periods, produce
antimicrobial substances, modulate immune responses and influence the metabolic
activities of the gut (Dunne et al., 1999). The properties of the strain should be well
documented (Dunne et al., 1999). Although some criteria, such as the non-pathogenic
status, technological suitability and careful documentation of the probiotic effects of a
microbial strain, are invariably required, no single strain is likely to carry all of the
abovementioned properties (Dunne et al., 1999). Moreover, probiotic properties are
considered strain-specific, and results obtained with one strain cannot therefore be
claimed for another, even a closely related strain. Microbes used in probiotic products
10
include strains from several Lactobacillus and Bifidobacterium species, Enterococcus
faecalis and Enterococcus faecium, Lactococcus lactis, Leuconostoc mesenteroides,
Pediococcus acidilactici, and Sporolactobacillus inulinus, Streptococcus thermophilus, as
well
as
Bacillus
cereus,
Escherichia
coli,
Propionibacterium
freudenreichii,
Saccharomyces cerevisiae and Saccharomyces boulardii (Holzapfel et al., 1998).
However, lactobacilli and bifidobacteria are most common in probiotic products designed
for human use; probiotic properties of these bacteria are also the best studied (Tannock,
1999b).
Lactobacilli belong to the lactic acid bacteria, which comprise a diverse group of
Gram-positive bacteria, most typically represented by non-sporing, catalase-negative,
devoid of cytochromes, non-aerobic but aerotolerant, fastidious and acid-tolerant cocci or
rods producing lactic acid as the major end-product during the fermentation of
carbohydrates (Axelsson, 1998). Being of fastidious nature, lactic acid bacteria require a
rich environment for growth, such as decaying plant material, food products and a
mammalian gastrointestinal tract or vagina. The genus Lactobacillus is heterogeneous,
containing species with 32-53% G+C of the chromosomal DNA arranged into three
groups based on differences in sugar metabolism caused by the presence or absence of
fructose-1,6-diphosphate aldolase and phosphoketolase (Axelsson, 1998). Although
possessing some phenotypical features common for lactic acid bacteria, the genus
Bifidobacterium is actually related to the Actinomycetes branch, having a high
chromosomal G+C content (Axelsson, 1998). Sugar metabolism of bifidobacteria differs
from that of lactic acid bacteria; the bifidobacteria lack aldolase and glucose-6-phosphate
dehydrogenase, and hexose sugars are exclusively degraded by the fructose-6-phosphate
pathway characterised by fructose-6-phosphate phosphoketolase (Ballongue, 1998).
Bifidobacteria are predominant members of the human intestinal microbiota, with
bacterial counts of 109-1011 per gram of stool, with B. bifidum, B. longum, B. infantis, B.
breve, B. adolescentis, B. angulatum, B. catenulatum, B. pseudocatenulatum, and B.
dentium reported as human isolates (Ballongue, 1998).
Several positive effects have been proposed for probiotic lactobacilli and
bifidobacteria. Antagonism towards intestinal pathogens has been demonstrated for
probiotics (Fernandes et al., 1987; Asahara et al., 2001; Fujiwara et al., 2001a, b, c).
11
Alleviation of diarrhoea is a well-documented characteristic of some strains (Bennet et
al., 1996; Kaila and Isolauri, 1996; Arvola et al., 1999; Orrhage et al., 2000; de Roos and
Katan, 2000; Van Niel et al., 2002), and particularly the ability of Lactobacillus
rhamnosus GG to shorten the duration of acute rotavirus diarrhoea has been established
(Kaila and Isolauri, 1996; de Roos and Katan, 2000). Stimulation of the gut immune
system by probiotic strains has been reported (Link-Amster et al., 1994; Kishi et al.,
1996; Miettinen et al., 1996 and 1998; Majamaa and Isolauri, 1997; Murosaki et al.,
1998; Pessi et al., 1999, 2000; Isolauri et al., 2000; Maassen et al., 2000), and induction
of cytokine profiles has been shown to be strain-dependent (Miettinen et al., 1996, 1998;
Maassen et al., 2000). Alleviation of allergic reactions in the gastrointestinal tract has
also been suggested for some strains: using a mouse model, Murosaki et al. (1998)
showed that heat-killed Lactobacillus plantarum L-137 suppressed production of antigenspecific IgE by stimulating the production of IL-12. In addition, Lactobacillus rhamnosus
GG and Bifidobacterium lactis Bb-12 have been demonstrated to relieve symptoms of
atopic eczema (Majamaa and Isolauri, 1997; Isolauri et al., 2000; Kalliomäki et al.,
2001). Administration of L. rhamnosus GG has been shown to enhance the production of
IL-10, acting thus as an anti-inflammatory mediator in atopic children (Pessi et al., 2000).
Consumption of probiotics has also been observed to lower activities of harmful faecal
enzymes (Goldin et al., 1992; Spanhaak et al., 1998). Some animal models have even
suggested the ability of probiotic strains to reduce the incidence of cancer (Pool-Zobel et
al., 1993; Goldin et al., 1996; Matsuzaki et al., 1996; Matsuzaki, 1998; O’Mahony et al.,
2001). Finally, probiotics may have significance for alleviation of intestinal disturbances.
For example, ingestion of a mixture of lactobacillar and bifidobacterial strains together
with a Streptococcus salivarius ssp. thermophilus was observed to help maintain
remission status in ulcerative colitis patients (Venturi et al., 1999).
Although lactobacilli and bifidobacteria are stated as GRAS (generally recognized
as safe) organisms due to their long usage and non-pathogenic status, safety issues have
been investigated. Isolation of these organisms from infections has been reported with a
low incidence, and in most cases, bifidobacteria and lactobacilli were associated with
immunocompromised patients (Brook, 1996; Saxelin et al., 1996). One major concern
among lactic acid bacteria is, however, resistance to vancomycin, especially within the
12
genus Enterococcus where the resistance has been shown to be transferable (Arthur et al.,
1996). Vancomycin resistance has been reported for lactobacilli by several authors
(Swenson et al., 1990; Herrero et al., 1996; Charteris et al., 1998; Zarazaga et al., 1999;
Felten et al., 1999; Klein et al., 2000), but it is generally considered to be an intrinsic
property. Vancomycin resistance expressed by the probiotic strain L. rhamnosus GG has
been studied in detail (Tynkkynen et al., 1998). Strain GG was not observed to transfer
vancomycin resistance or receive other resistance elements from enterococci, nor were
any genes resembling enterococcal vancomycin resistance genes found in Lactobacillus
GG (Tynkkynen et al., 1998). Similarly, Klein et al. (2000) reported no indications for
the presence of the vanA gene cluster, the vanB gene or the vanC gene from five L.
reuteri strains or L. rhamnosus GG, suggesting that the vancomycin resistance of the
strains studied is unrelated to the acquired resistance in the Enterococcus species.
2.3. Genetic labelling of lactic acid bacteria
Insertion of an extra DNA label into a target strain genome provides a way to
monitor the specific strain in various environments. However, introduction of foreign
DNA is often required, and in many cases, genetic elements producing a phenotypic
change are used to distinguish the marked strain. Genetic elements encoding resistance to
an antibiotic have been utilised for labelling of lactic acid bacteria (Rush et al., 1994,
Klijn et al., 1995), but the increasing spread of antibiotic resistance factors between
bacterial species or genera make such approaches unsuitable for strains intended for
human or animal use. Therefore, usage of various ‘food-grade’ markers has been
suggested. Labelling of lactic acid bacteria with a plasmid-encoded green fluorescent
protein gene placed under an inducible promoter has been reported for Lactococcus lactis
and Lactobacillus plantarum (Geoffroy et al., 2000), whereas Allison and Klaenhammer
(1996) suggested the use of a native Lactobacillus gene encoding immunity to Lactacin F
as a food-grade genetic marker. A similar role has also been demonstrated for ltnI
conferring immunity to lacticin 3147 (McAuliffe et al., 2000). Site-specific integration of
desired genetic elements into bacterial chromosomes through phage attachment sites has
been described for Lactococcus lactis (Lillehaug et al., 1997), Lactobacillus delbrueckii
and Lactobacillus plantarum (Dupont et al., 1995).
13
Legislation as well as consumer acceptance limit the usage of genetically
engineered organisms, and therefore, genetic changes introduced to target organisms
must be carefully considered. Maguin et al. (1996), for example, combined insertion
sequence ISS1 with a thermosensitive replicon, enabling a high frequency of random
insertion (about 1%) with Lactococcus, Enterococcus and Streptococcus thermophilus,
while efficient excision of the plasmid generated stable mutants with no foreign markers,
leaving only a single ISS1 copy at the mutated site. However, the smallest detectable
alteration introduced to a target organism is changing one or a few bases in a gene-coding
sequence without affecting the amino acid sequence of the corresponding gene product.
In principle, such a mutation could be caused by natural processes due to the degeneracy
present in the genetic code. Silent mutations have been utilised for modification of a
Lactococcus lactis subsp. cremoris strain plasmid-encoded proteinase prtP gene by in
vitro mutation of the third positions of four adjacent codons, thus providing a genetic
label with no phenotypic effects (Hertel et al., 1992). Although technically more
demanding, genetic marking should, however, preferably be directed to gene replacement
on a chromosomal locus to ensure maximal stability of the alterations introduced.
2.4. Utilisation of nucleic acid based methods for identification and
monitoring of bacteria in population samples
2.4.1. Utilisation of methods independent of prior knowledge on sequence data
2.4.1.1. Sequencing analysis of 16S ribosomal DNA populations
Cloning and sequencing of the 16S ribosomal DNA (rDNA) pools in a population
sample provides a method for obtaining sequence-level information on uncultivable
bacteria abundantly present in various parts of the gastrointestinal tract. Suau et al. (1999)
analysed the sequence of 284 16S rDNA clones derived from one faecal sample and
classified the clones into 82 molecular species, using 98% similarity criteria for a species.
Importantly, only 24% of the molecular species were derived from a described organism
(Suau et al., 1999). Similarly, comparative 16S rDNA sequence analysis of the intestinal
bacterial community in pigs revealed only a 17% fraction of previously described
14
organisms among a total of 375 phylotypes (Leser et al., 2002). In a large-scale study of
subgingival plaque samples, including analysis of a sequence of 2522 16S rDNA clones,
347 species was observed among the clones studied, which correlated well with a
previous estimate of expected species diversity in the oral cavity (Paster et al., 2001).
Thus, the direct cloning approach seems to give a good idea of the total microbiota
present in a complex population. It also facilitates determination of species-level
differences between bacterial populations, as shown by the discovery of novel phylotypes
and species not previously associated with childhood caries by comparing of the oral
microbiota of a healthy subject and a subject with early childhood caries (Becker et al.,
2002).
Approaches based on cloning are, however, rather tedious and are not optimal for
analysis of large numbers of samples. Like all PCR-dependent methods, construction of
clone libraries may be prone to biasing, possibly leading to falsification of the library
structure (Bonnet et al., 2002). Nevertheless, with the help of cloning, design of
phylogenetically relevant oligonucleotide probes is enabled (Suau et al., 1999).
2.4.1.2. Denaturing gradient gel electrophoresis (DGGE) and thermal gradient
gel electrophoresis (TGGE)
Denaturing gradient gel electrophoresis (DGGE) and thermal gradient gel
electrophoresis (TGGE) have become popular methods for analysis of microbial
populations present in various habitats such as water ecosystems (Overmann et al., 1999),
microbial fermentations (Ampe et al., 2001), and the GI tract (Millar et al., 1996;
Zoetendal et al., 1998; Simpson et al., 2000; McCracken et al., 2001; Satokari et al.,
2001a, 2001b; Walter et al., 2001; Favier et al., 2002; Heilig et al., 2002; Requena et al.,
2002; Zhu et al., 2002). These methods allow separation of nucleic acid molecules based
on their size and sequential differences. Thus, a population of DNA molecules, such as
the 16S ribosomal RNA (rRNA) or PCR-amplified 16S rDNA, can be studied and
predominant members of the population identified via sequencing of isolated nucleic acid
bands. Sensitivity of gradient gel electrophoresis is affected by the choice of PCR primers
used. For example, utilisation of universal 16S primers limits the sensitivity to detection
of 1% subpopulations (Zoetendal et al., 1998). By contrast, utilisation of species- or
15
group- specific primers may allow detection and identification of bacteria representing a
minority of the total population (Walter et al., 2001; Heilig et al., 2002).
Drawbacks of gradient gel electrophoresis include difficulty of comparisons
between individual gels, requirement of careful adjustments, and the need for sequencing
to confirm identities of bands seen in a gel. Furthermore, some bacteria may remain
unidentified due to low resolution of DNA bands (Ampe et al., 2001). With both DGGEand TGGE-based methods, sequencing is required for correct identification of individual
bands seen in the gel. As stated by Schmalenberger et al. (2001), intraspecies operon
heterogeneities may significantly contribute to genetic profiles in microbial community
analysis, as amplification of one bacterial DNA may yield several separate bands, which
can then be wrongly interpreted as high microbial diversity. Depending on the 16S
binding universal primer pairs used, single-strand conformation polymorphism analysis
(SSCP) revealed an average of 1.7 – 2.3 bands per pure cultured bacterial organism
(Schmalenberger et al., 2001).
2.4.1.3. Terminal restriction fragment length polymorphism (T-RFLP)
Terminal restriction fragment length polymorphism (T-RFLP) is based on
endonuclease digestion of PCR-amplified DNA and capillary electrophoresis analysis of
the terminal restriction fragment (TRF) containing a fluorescent label. Terminal
restriction patterns have been used to analyse marine bacterioplankton communities
(Moeseneder et al., 1999, 2001) as well as faecal bacteria (Kaplan et al., 2001). The
method is, however, limited by the choice of primers, which can, with their different
affinities, dramatically change the patterns observed, while another problem is the TRF
length overlap by phylogenetically distant bacteria (Kaplan et al., 2001).
2.4.1.4. Analysis of community DNA profiles
Analysis of total bacterial community structure can be accomplished by
measurement of guanosine-cytosine profiles of a population DNA sample with DNA
reassociation (Griffiths et al., 1997; Ritz et al., 1997; Chatzinotas et al., 1998) and density
gradient centrifugation (Holben and Harris, 1995; Apajalahti et al., 1998; Nüsslein and
16
Tiedje, 1998; Apajalahti et al., 2001). Density gradient fractionation of community DNA
enables analysis of interesting fractions by cloning of PCR-amplified DNA pools,
followed by sequencing (Nüsslein and Tiedje, 1998; Apajalahti et al., 2001). Density
gradient centrifugation has been applied in analysis of intestinal microbial guanosinecytosine profiles (Apajalahti et al., 1998, 2001). However, because this approach requires
sophisticated and expensive equipment, it is out of reach for most research laboratories.
2.4.2. Utilisation of specific oligonucleotide primers or probes
2.4.2.1. Hybridisation
Hybridisation offers a means for direct semi-quantitative monitoring of
population samples. A very high sensitivity for detection of DNA targets can be obtained
with radioactively labelled probes (Palva, 1985). However, because of its abundance in
bacterial cells, rRNA provides for a more attractive target for hybridisation studies.
Indeed, hybridisation assays targeting rDNA have been verified as 10-fold less sensitive
than assays for rRNA (Muttray and Mohn, 2000). Dot blot hybridisation with rRNA –
targeted probes has been used for semiquantitative analysis of ruminal microbes (Forster
et al., 1997) and intestinal microbiota (Doré et al., 1998; Sghir et al., 2000; Hopkins et al.,
2001). Comparison of hybridisation results of a specific probe with a universal probe
enables assessment of the target bacterial proportion present in a sample (Doré et al.,
1998; Sghir et al., 2000; Hopkins et al., 2001; Marteau et al., 2001). At best, detection of
a 0.1 – 0.01% rRNA subpopulation has been reported (Marteau et al., 2001),
corresponding to approximately 107 target cells if 1011 bacteria are considered to be
present in a gram of faeces. Use of radioactive isotopes, especially in large-scale and
long-term analyses, is, however, complicated by the short half-lives of labels. Nonradioactively labelled probes are more convenient, but their sensitivity is limited. Preenrichment of the target DNA by polymerase chain reaction can be used to enrich the
target DNA. For example, 0.1 pg of B. distasonis or B. thetaiotaomicron DNA
(approximately 10-20 cells) or 0.01 pg of B. vulgatus DNA (1-2 cells) applied to a PCR
was sufficient to produce a positive hybridisation signal with specific digoxigeninlabelled probes (Kreader, 1995).
17
An additional factor of concern for filter hybridisations is the need to label each
probe separately and to perform hybridisation reactions after optimisations in separate
vessels. However, Ehrmann et al. (1994) described a reverse dot blot hybridisation with
several membrane-bound oligonucleotide probes for identification of lactic acid bacteria
present in mixed populations. Similarly, Becker et al. (2002) used simultaneous detection
of PCR-amplified rDNA of 23 oral bacterial species or groups with oligonucleotide
probes.
In the fluorescence in situ hybridisation (FISH) method, the bacterial cell samples
to be studied are immobilised on microscope slides and made permeable for fluorescently
labelled oligonucleotides with subsequent microscopic observation of the hybridisation
signal intensities. FISH has been used for monitoring faecal microbiotas (Langendijk et
al., 1995; Franks et al., 1998; Harmsen et al., 2000a, 2000b, 2002; Jansen et al., 1999;
Schwiertz et al., 2000), and detection of a 1 – 0.1% bacterial subpopulation has been
obtained (Schwiertz et al., 2000; Harmsen et al., 2002). Automated analysis of
fluorescent signals has also been utilised to facilitate objective interpretation of results
(Jansen et al., 1999). The major problem with FISH applications is the different
penetration of probes in bacteria with various cell wall types, resulting in a possible
underestimation of Gram-positive bacteria (Langendijk et al., 1995; Jansen et al., 1999).
4.4.2.2. Polymerase chain reaction (PCR)
Amplification of target nucleic acids with PCR is an effortless method for
detection of target DNA from various samples. In principle, the detection limit for a PCR
assay, based on usage of one or two oligonucleotide primers specific for the target
bacteria, is the presence of one copy of the amplified DNA region. Although achievement
of such an extremely sensitive assay is unlikely, the PCR remains a very powerful
detection method, typically requiring a minimum of 10-20 target copies for successful
amplification. The high sensitivity is also somewhat problematic, as a simple aerosol
contamination may lead to false amplification.
PCR-based detection of rRNA genes has been used for direct detection of various
bacteria present in faeces (Kok et al., 1996; Wang et al., 1996, 1997; Lawson et al., 1998;
Matsuki et al., 1999; Bernhard and Field, 2000; Saito et al., 2002) and pathogens from
18
clinical samples (Harper-Owen et al., 1999). Partial or full amplification of the 16S
rDNA with primers specific for a genus or a few closely related genera has also been
reported (Kok et al., 1996; Kaufmann et al., 1997; Walter et al., 2001; Heilig et al.,
2002). In addition, utilisation of 16S-23S intergenic sequences as targets of speciesspecific primers for different lactic acid bacteria has been described (Tilsala-Timisjärvi
and Alatossava, 1997). Good sensitivities have been reported for the PCR detection
assays of faecal bacteria. For example, PCR detection sensitivity of five ruminococcal
species with species-specific primers from faecal samples spiked with the target species
did not markedly differ from the observed detection limit of 4-100 cells from pure
cultures (Wang et al., 1997).
Although a highly potent detection method, results obtained by conventional endpoint PCR should not be considered to be directly quantitative. PCR may lead to
differential amplification of target templates originally present in equal amounts
(Reysenbach et al., 1992). In addition, very low template concentrations may generate
random fluctuations in priming efficiency of population DNA samples with universal
primers, leading to a bias in the end-product concentration (Chandler et al., 1997).
Knowledge of the rRNA gene copy numbers and genome sizes of bacteria in a mixed
DNA sample has been observed to be insufficient in predicting the final product ratio of a
PCR amplification (Farrelly et al., 1995). Suzuki and Giovannoni (1996) also noticed that
the accumulation of end products during mixed-template PCR caused biasing of the
various end-product ratios towards a 1:1 situation, which was hypothesised to be caused
by an increase in the homologous template hybridisation, decreasing the efficiency of
primer annealing and subsequent amplification.
Some improvements in the reliability of PCR quantification have been obtained
by competitive PCR approaches. Hahn et al. (1995) created a quantitative PCR assay
based on post-amplification differentiation of the internal standard and the sample DNA
by selective restriction analysis and digoxigenin-based colorimetric detection.
Quantitative detection of Mycobacterium tuberculosis PCR products was performed by
enzyme-linked immunosorbent assay by comparison of hybridisation results with two
probes and two IS6110 elements derived from either an internal control or a modified
template (Kox et al., 1996). With co-amplification of an internal standard, detection of
19
Clostridium proteoclasticum was linear between 1 × 104 and 5 × 101 cells, with a
detection limit of 50 fg or 25 cells (Reilly and Attwood, 1998), whereas detection of
Oxalobacter formigenes from human faecal samples by competitive PCR was shown to
be linear over a range of six logarithmic units, with a detection limit of approximately
100 genomes (Sidhu et al., 1999).
2.4.2.3. Polymerase chain reaction – enzyme-linked immunosorbent assay
(PCR-ELISA)
Polymerase chain reaction – enzyme-linked immunosorbent assay (PCR-ELISA)
combines utilisation of polymerase chain reaction for efficient multiplication of the target
DNA and hybridisation with a detection probe to ensure the specificity of the reaction.
Figure 1 summarises the concept of PCR-ELISA detection. The PCR-amplified products
are labelled with digoxigenin during or after the amplification reaction and hybridised
with the specific biotinylated detection probe. The probe is immobilised in streptavidincoated microtitre plate wells, and hybridised DNA products are detected via digoxigenintargeted antibodies linked with an enzyme capable of producing a colorimetric or
fluorimetric signal when brought together with a substrate. Usage of PCR-ELISA
hybridisation for detection of single pathogens of clinical importance and spoilage
bacteria of food has been described (Hahn et al., 1995; Kox et al., 1996; Denis et al.,
1997; Gutiérrez et al., 1997; Nelson et al., 1997; Bazzichi et al., 1998; Fletcher et al.,
1998; Gutiérrez et al., 1998; Löffler et al., 1998; Beyer et al., 1999; González et al., 1999;
Fischer-Romero et al., 2000; Garcia et al., 2001; Sails et al., 2001). In comparison with
filter hybridisation, PCR-ELISA provides more easily standardised reaction conditions by
utilisation of commercially supplied microtitre plates. Other advantages of PCR-ELISA
compared with several other methods are its relative simplicity and low costs.
20
Figure 1. PCR-ELISA detection. The PCR-amplified, digoxigenin-labelled target molecule is hybridised
with a specific probe captured into a microtitre well via biotin-streptavidin linkage. After removal of
unbound DNA by washing, the hybridised molecules are visualised with the help of anti-digoxigenin
antibodies conjugated with horse radish peroxidase (POD). With the application of 3´,3,5´,5tetramethylbenzidine (a substrate for POD), a blue colour is produced by the peroxidase. The reaction is
terminated with sulphuric acid, after which the colour produced can be measured at 450 nm.
Good sensitivities have been reported for PCR-ELISA assays, and the results can
in some cases be interpreted quantitatively. The limit for detection of Bordetella pertussis
was 100 target organisms, starting from appliance of different target DNA amounts to
PCR reaction (Nelson et al., 1997). Less than ten Epstein-Barr virus genome copies
added to 750 ng of background DNA were required for a positive PCR-ELISA result
(Bazzichi et al., 1998). Similarly, a sensitivity of 5 cfu/ml blood was obtained for
detection of C. albicans and A. fumigatus cells, corresponding to the sensitivity of
Southern blotting using digoxigenin-labelled oligonucleotides (Löffler et al., 1998).
21
Fletcher et al. (1998) could detect Aspergillus fumigatus quantitatively by a PCR-ELISA
method on a log-scale between 100 and 1 pg of target DNA. Detection of Escherichia
coli in oysters was reported to be quantitative in the range of 10-105 cfu/g (González et
al., 1999). Detection of Campylobacter jejuni and C. coli with PCR-ELISA was shown to
be 10- to 100-fold more sensitive than a gel-based PCR method using the same primers,
the smallest amount of C. jejuni template DNA giving a positive signal in the assay being
1.5 fg (Sails et al., 2001). Some of the described PCR-ELISA applications can be
considered to be quantitative competitive PCR approaches (Hahn et al., 1995; Kox et al.,
1996; Denis et al., 1997).
2.4.2.4. Real-time PCR
Real-time PCR is based on on-line measurement of the amplification reaction,
enabling quantification of the product during the logarithmic phase of PCR. The first and
so far most commonly used real-time PCR approach is the 5´-nuclease (TaqMan) assay
introduced by Holland et al. (1991). The original assay operates using a radioactively
labelled probe that hybridises in the PCR template region to generate a specific,
detectable signal from the amplification reaction. The detection probe becomes annealed
to one of the DNA strands during the amplification and is cleaved by the Thermus
aquaticus DNA polymerase 5´-exonuclease activity during the primer extension step
(Holland et al., 1991). Lee et al. (1993) further extended the 5´-nuclease assay by
utilisation of doubly labelled oligonucleotide probes for fluorescent measuring of
formation of the specific PCR product during primer extension (Figure 2). Introduction of
an automated detection method enabled real-time monitoring of the PCR product
formation during the exponential amplification phase (Heid et al., 1995). Since the advent
of real-time PCR, several techniques have been introduced, including primers with
fluorescent dyes, molecular beacons, dual probes and intercalating dyes such as SYBR
Green I. Real-time PCR is a superior technique for quantification of nucleic acids. While
competitive PCR has been demonstrated to be as reproducible and accurate as real-time
PCR, the latter has the benefit of an easier methodology, reducing the need for sample
DNA treatment (Desjardin et al., 1998; Wall and Edwards, 2002).
22
Figure 2. 5´-nuclease assay. A dual-labelled oligonucleotide probe is designed to hybridise near the 3´-end
of one of the primers used in the amplification reaction. An intact probe contains two fluorescent
molecules, and at its 5´-end the reporter dye is quenched due to its near vicinity to the quencher dye at the
3´-end. Upon primer annealing, the probe is digested by the 5´-nuclease activity of the DNA polymerase.
This results in separation of the two fluorescent molecules. Measurement of light (λ2) emitted by the
reporter is now enabled.
Although real-time PCR is a relatively new technique, several detection or
quantification assays targeting various bacteria have already been described. Target
bacteria include carious dentine bacteria (Martin et al., 2002), Desulfotomaculum from
soil (Stubner, 2002), faecal bifidobacterial species (Requena et al., 2002), Helicobacter
hepaticus (Ge et al., 2001), Staphylococcus aureus (Hein et al., 2001), Borrelia
burgdorferi sensu lato (Rauter et al., 2002), Campylobacter jejuni (Nogva et al., 2000a),
Listeria monocytogenes (Nogva et al., 2000b), Rhodococcus coprophilus (Savill et al.,
2001) and Mycoplasma genitalium (Yoshida et al., 2002). Some applications for
determination of larger bacterial groups have also been published (Lyons et al., 2000;
Suzuki et al., 2000; Bach et al., 2002; Nadkarni et al., 2002). Typically, sensitivities of 1
to 100 target genomes per reaction and linearity ranges of 4 to 8 logarithmic units are
obtained in assays targeted to specific bacteria. Real-time PCR is also utilised as a rapid
23
diagnostic tool for detection of pathogenic bacterial species or strains present in a sample.
With pathogenic bacteria, the target of choice for real-time PCR is generally a gene
associated with the pathogenic traits (Ke et al., 2000; Bellin et al., 2001; Corless et al.,
2001; Ge et al., 2001; Hein et al., 2001; Bélanger et al., 2002; Rauter et al., 2002;
Tondella et al., 2002).
Figure 3. SYBR Green I –based measurement of amplification products. SYBR Green I is an
intercalating dye with virtually no fluorescence unless bound to double-stranded DNA. During PCRamplification formation of double-stranded DNA molecules can be measured as an increase in SYBR
Green I fluorescence.
Detection of PCR amplicons with specific probes is often favoured over usage of
intercalating dyes due to the former’s better sensitivity and lack of detection of falsely
primed products. However, SYBR Green I (Figure 3) has become popular because of the
possibility to use this intercalating dye in virtually any assay. Real-time quantitative PCR
with SYBR Green I has been reported to be 10-fold less sensitive than a corresponding
TaqMan assay due to the formation of non-specific products in reactions starting with
small amounts of template DNA (Hein et al., 2001). On the other hand, SYBR Green I
has great potential in situations where a diverse target population is to be detected with
24
PCR (Bach et al., 2002). A probe-based methodology requires a binding site for the probe
in the vicinity of one of the primers; however, such a conserved site is likely to be
missing from a degenerate target DNA population. This should be taken into account,
especially when a bacterial population containing several hitherto unknown species is
studied.
25
3. Aims of the study
The aims of this study were to develop molecular methods for analysis and monitoring of
faecal bacterial populations and putative probiotic strains as well as to characterise the
technological properties of two Lactobacillus brevis strains. The following goals were
set:
1.
To create a genetic label by introducing silent mutations to adjacent amino
acid codons of a genomic peptidase gene of a selected Lactobacillus strain and
to confirm the unchanged phenotype of the strain with an available peptidase
assay.
2.
To develop oligonucleotide PCR primers or probes targeting the 16S
ribosomal DNA as species- or group-specific detection tools.
3.
To exploit a PCR-ELISA application with multiple oligonucleotide capture
probes for analysis of artificial mixed DNA samples or faecal DNA
preparations.
4.
To test suitability of real-time PCR for quantification of selected intestinal or
probiotic bacteria in faecal samples.
5.
To evaluate the applicability of two Lactobacillus brevis strains as
supplementary strains with potential probiotic actions in dairy products.
26
4. Materials and methods
4.1. Microbial strains, plasmids, human cell lines and culture conditions
The microbial strains, plasmids and human cell lines used in this study have been
described in detail in the respective original publications I-V. Briefly, Lactobacillus
helveticus CNRZ32 was chosen as a model organism for demonstration of genetic
labelling (I). Plasmids pUC19 (Yanisch-Perron et al., 1985) and pSA3 (Dao and Ferretti,
1985) were used to construct plasmids pKTH5052 and pKTH5053, respectively (I). The
PCR-ELISA application with universal primers targeting 16S and 23S rDNA was tested
with a set of bacteria including 25 species, that represented type strains of lactobacilli,
bifidobacteria and other bacteria reported as members of human intestinal microbiota, or
lactobacilli species used in dairy fermentations (II). Extension of the PCR-ELISA method
with bifidobacteria-targeted 16S rDNA amplification was tested with a set of ten
bifidobacterial species belonging to the human intestinal microbiota, suggested as
potentially harmful oral microbes, or used in dairy products (III). With real-time PCR, six
bacterial strains, each representing a bacterial species or group, present in the human
intestinal tract or dairy products, were used (IV). Dairy technological and probiotic
properties of two lactobacilli strains, L. brevis GRL1 (ATCC 8287) and L. brevis GRL62
(ATCC 14869T), were determined using dairy starters, positive or negative control strains
or target strains for testing the antagonistic properties (V). The human intestinal cell lines
Intestine-407 and Caco2 were used in adhesion studies (V). Culturing of microbes and
human cell lines was carried out using media and conditions as outlined in the Materials
and methods sections of original publications (I-V).
4.2. Basic DNA techniques
Rapid isolation of genomic DNA from pure cultured bacteria was performed by
cell mill disruption of bacterial samples in the presence of glass beads, followed by
phenol-chloroform extraction and ethanol precipitation (I, II, III, V). For rapid and
effective purification and isolation of DNA from faecal material, a method described in
Study I was used (I, III). A large-scale DNA isolation method described by Apajalahti et
27
al. (1998) was used to produce a sufficient amount of template DNA for real-time PCR
(IV).
Wizard Minipreps (Promega, USA) were used for isolation of plasmid DNA from
E. coli clones (I), whereas the Qiagen Plasmid Protocol Kit (Qiagen, Hilden, Germany)
was exploited for isolation of plasmid DNA from L. brevis (V).
Restriction enzyme digestions and ligations were carried out according to the
enzyme manufacturer’s recommendations (Promega). The transformations of L.
helveticus cells were performed as described by Bhowmik and Steele (1993). Dot blot
hybridisation was executed using standard methods (IV). DNA concentrations were
measured with a Versafluor fluorometer (Bio-Rad). DNA sequencing was performed
with an ABI310 DNA sequencer (Applied Biosystems) using BigDye Terminator
chemistry (I).
4.3. Design of oligonucleotide primers and probes
Oligonucleotide primers and probes were designed with the help of published
sequence data available in sequence databanks (I, II, III, IV). Generally, the online
Internet tools ClustalW (Thompson et al., 1994) and Fasta3 (Pearson and Lippman, 1988)
provided by the European Bioinformatics Institute (EBI) were used for identifying the
primers and probes with desired specificity towards intended target DNA.
Oligonucleotides required for creation of the silent mutation site (I) were planned by
utilising the sequence of the L. helveticus CNRZ32 pepX gene (Accession number
U22900). While creating the mutations, major changes in the codon usage frequency
were avoided to reduce the likelihood of changes in the expression level of the target
gene. The Ribosomal Database Project (Maidak et al., 2001) was utilised for designing
the primers and probes used in Study IV. In addition, signature oligonucleotides
published previously by others were utilised or modified when necessary (II, III, IV).
4.4. Polymerase chain reaction (I-V)
For end-point detection or production of DNA templates, polymerase chain reactions
were carried out in reaction conditions recommended by the manufacturer of Dynazyme
28
DNA polymerase (Finnzymes). In Study I, the PCR conditions used in detection of wildtype and mutant strains were as follows: the reaction mixture consisted of 50 mM TrisHCl pH 9.0, 15 mM (NH4)2SO4, 0.1% Triton X-100, 1.8 mM MgCl2, 360 µM of each
deoxynucleotide triphosphate, 1 µM of each primer and 0.02 U/µl Dynazyme EXTTM
Polymerase (Finnzymes, Finland), and 1 µl of template or water. Each sample was
subjected to a primary denaturation cycle at 95ºC for 2 minutes followed by 30 cycles of
denaturation at 95ºC for 30 seconds, annealing at 55ºC for 30 seconds and elongation at
72ºC for 1.5 minutes. The reaction was terminated at an elongation step for 5 minutes at
72ºC and followed by incubation at 4ºC. In Study IV, gradient PCR was used to select
optimal annealing temperatures for PCR primer pairs. Oligonucleotides were synthesised
by commercial suppliers.
4.4.1. PCR-ELISA (II, III)
PCR-ELISA was carried out as described in Studies II and III. Briefly, the PCR
products were labelled with the DIG-High Prime Kit (Boehringer Mannheim, Germany)
according to the instructions of the manufacturer, and concentrations of the PCR products
were measured with the Versafluor fluorometer. For calculation of molecular quantities
in the labelled end products, the amount of DNA (ng/µl) was divided by the length of the
PCR product (base pair) multiplied by the average weight of a base pair (ng/base pair).
The hybridisations were performed in commercial streptavidin-coated microtiter plates
(Labsystems, Finland). Results were analysed by measuring absorbance at 450 nm (iEMS
Reader MF, Labsystems, Finland).
4.4.2. Real-time PCR (IV)
Real-time PCR was tested with two different chemistries (5´-nuclease- and SYBR
Green I assays) as described in Study IV. Quantitative PCR was performed with an
iCycler iQ (Bio-Rad, USA) and iCycler Optical System Interface software version 2.3
(Bio-Rad, USA). All PCR reactions were performed in triplicate, in a volume of 25 µl,
using 96-well optical grade PCR plates and optical sealing tape (Bio-Rad, USA).
29
Optimal concentrations for various reaction components were tested for each
primer set and chemistry with a dilution series of genomic DNA from the target test
species. With SYBR Green I chemistry, effect of the polymerase type on PCR product
formation was tested with a standard polymerase, Dynazyme II (Finnzymes, Finland),
and hot-start polymerases AmpliTaq Gold® DNA Polymerase (Applied Biosystems,
USA), BlueTaq (Euroclone Ltd., UK) and FastStart Taq DNA Polymerase (Roche). The
TaqMan assays were performed successfully with the Dynazyme II enzyme and therefore
switching to the hot-start enzyme was not considered.
4.5. Peptidase activity assays (I)
Activity measurement of the PepX was determined from both the wild-type and
mutated L. helveticus strains according to the method of El Soda and Desmazeaud
(1982).
4.6. Evaluation of L. brevis strains as dairy adjuncts
In vitro tolerance of the L. brevis strains to low pH, bile salts and pancreatic fluid
was examined as described in Study V. Antimicrobial properties towards selected food
spoilage bacteria and dairy lactic acid bacteria starters were evaluated by following the
microbial growth in MRS broth supplemented with 10% filter-sterilized L. brevis GRL1
or GRL62 supernatant at 37ºC for two days with an automated turbidometer Bioscreen C
Analysing System (Labsystems, Finland). Adherence to Caco-2 and Intestine-407 cell
lines was studied as described in Study V. Resistance to selected antibiotics was tested
with disc diffusion and microdilution methods, modified from instructions given by the
antibiotic disc manufacturer (A/S Rosco, Denmark) and NCCLS. In vivo feeding trials
with four healthy volunteers were performed to see whether the L. brevis strains could
survive the GI passage. Technological suitability of the strains for dairy processes was
also evaluated as described in Study V.
30
5. Results
5.1. Strain-specific genetic labelling of Lactobacillus helveticus CNRZ32 (I)
Silent mutation positions were introduced in adjacent codons of the Lactobacillus
helveticus CNRZ32 chromosomal pepX gene without altering the amino acid sequence of
the gene product, creating a strain-specific tag which enabled direct nucleic-acid level
identification of the changed strain (Figure 4). The mutation site was introduced into the
pepX gene with a PCR primer containing designed mismatched nucleotides. The target
gene was amplified in two separate reactions to obtain a conserved fragment and a
mutation site-containing fragment, which were ligated together to form a single mutation
site-containing fragment. The mutant gene was first cloned with pUC19 to E. coli to
create plasmid pKTH5052 and further subcloned to pSA3, resulting in the plasmid
pKTH5053, which was then used to transform L. helveticus CNRZ32.
Figure 4. Schematic representation of the silent-mutation site –containing pepX gene.
A L. helveticus CNRZ32 transformant strain GRL1021 containing the plasmid
pKTH5053 was grown at a restrictive temperature (45ºC) under antibiotic selection to
find clones with the thermosensitive plasmid integrated into the bacterial chromosome
through homologous recombination between the wild-type and mutant forms of pepX. An
erythromycin-resistant strain, observed to contain the integrated plasmid, was grown at
permissive conditions (37ºC) for approximately 100 generations to enable a second
31
homologous recombination event between the two pepX genes, resulting in excision of
the plasmid and one of the pepX genes. Samples were plated out and colonies that had
lost antibiotic resistance were screened with PCR for replacement of the wild-type pepX
with the mutated pepX. A strain with the mutated gene was found and designated as
GRL1023. Sequencing was used to confirm the correctness of the integration construct.
The intact phenotypic properties of the GRL1023 were verified by measuring the
activity of the target gene product PepX by utilising its ability to break down L-glycyl-Lprolyl-p-nitroanilide, with production of colour. End-point PCR with PCR primers
targeting the mutated or original pepX sequence was successfully used to detect the wildtype and mutant strains added to faeces or milk.
5.2. Suitability of PCR-ELISA for analysis of mixed DNA populations (II, III)
5.2.1. PCR-ELISA with universal primers targeting the 16S and 23S genes (II)
A PCR-ELISA method was tested with 16 oligonucleotide probes to
simultaneously detect under standardised conditions selected intestinal bacteria,
lactobacilli
and
bifidobacteria.
For
this
purpose,
species-
or
group-specific
oligonucleotide probes for lactobacilli and selected intestinal bacteria were designed or
adapted from the literature. Specificity of the probes was tested with 25 species. The 16S
and 23S region of these test species was first PCR-amplified with universal eubacterial
primers, followed by digoxigenin-labelling of the PCR products and addition of 2 × 1010
labelled molecules to each hybridisation reaction. For hybridisation, the oligonucleotide
probes to be tested were bound to the microtiter plate wells via biotin-streptavidin
linkage. Under the hybridisation conditions used, the specificity level expected was
obtained with most of the oligonucleotide probes chosen. Only the differentiation of
closely related L. casei, L. paracasei and L. rhamnosus was partly unsatisfactory.
Furthermore, in addition to its intended target DNA, the F. nucleatum probe, fuso16S,
also recognized E. biforme. Sensitivity of the PCR-ELISA with the probes tested varied
between detection of 6.77 × 107 and 1.29 × 109 PCR-amplified molecules in a
hybridisation reaction. The sensitivity obtained was somewhat lower than expected,
32
which was at least partly due to compromises that had to be made in the hybridisation
conditions when using several probes simultaneously.
PCR biasing was studied by combining genomic DNA from different test species
into two mixed sample pools, each with seven DNA targets, prior to PCR amplification.
PCR was also performed using the pools in the same amplification. The amplification
products were hybridised with 14 different probes. Most of the probes tested performed
well and could detect their target DNA from mixed samples of both seven and fourteen
species, but the probes for B. adolescentis (ado440, b162), L. brevis (lab86), L. paracasei
(par160), L. plantarum (pla448) and L. curvatus (cur150) failed to recognize their target
DNAs. Failure in the detection of B. adolescentis, L. paracasei and L. plantarum in
particular could have been due to either bias during multi-template PCR, where some
templates are amplified more efficiently as a result of differences in the specificity of
eubacterial primers, or bias by chance. Changing of the total template concentrations
tested in the PCR amplification was not observed to affect the hybridisation results.
5.2.2. PCR-ELISA with bifidobacterial primers targeting the 16S genes (III)
The PCR-ELISA method was extended for detection of the most common
Bifidobacterium species in humans and applied to a feeding trial including administration
of Bifidobacterium lactis Bb-12 and galacto-oligosaccharide -containing syrup as
probiotic and prebiotic preparations, respectively. Oligonucleotide probes based on 16S
rDNA sequences were designed and tested for specificity and sensitivity with nine
different bifidobacterial species, followed by analysis of faecal samples. Faecal
bifidobacteria were monitored for fluctuations during and after the feeding trial. Although
the bifidobacterial populations present in faeces were generally consistent between
samples taken from different time points, PCR-ELISA results suggested that
Bifidobacterium longum was partly replaced by B. lactis Bb-12 during the probiotic
feeding. This proposition could not, however, be verified with statistical methods.
The occurrence of bifidobacterial species or groups in human faecal samples is
summarised in Table 1. Generally, the bifidobacterial species composition of individual
33
subjects was consistent and comprised 3-4 bifidobacterial species or groups. The
predominant species were Bifidobacterium longum and B. adolescentis.
TABLE 1. Distribution of different bifidobacterial species in human subjects.
Species
No. of positive subjects
% of total
Bifidobacterium adolescentis
23
82.1
B. bifidum
12
42.9
4
14.3
B. catenulatum group
11
39.3
B. denticolens
0
0
B. dentium
0
0
B. lactis2)
0
0
B. longum
28
100
one species
1
3.6
two species
9
32.1
three species
13
46.4
four species
5
17.9
B. breve
1)
Combinations of
1)
B. catenulatum group consists of B. catenulatum and B. pseudocatenulatum
2)
Results are based on analysis of pre-feeding samples (0-samples)
5.3. Comparison of real-time PCR and dot blot hybridisation for
quantification of the 16S ribosomal DNA of target bacteria (IV)
Real-time PCR was tested with two chemistries: SYBR Green I and TaqMan
probes. Six detection assays were tested for their sensitivity in detecting target bacterial
DNA from pure and mixed samples with both chemistries. The same target DNAs were
also quantified with dot blot hybridisation probes. Real-time PCR was shown to have
sensitivity superior to dot blot hybridisation. The linear range of amplification of pure
cultured target DNA varied between 0.1-1 pg and 1-10 ng of specific target genome,
which corresponds to approximately 20-400 to 2×105–4×106 genomes, depending on the
genome size of the target species. Furthermore, a 1 pg addition of target DNA to 20 ng of
mixed DNA samples could be detected reliably with both real-time PCR chemistries. A
34
reconstruction assay with addition of 109, 108, 107, 106 or 105 target bacterial cells to
faecal samples confirmed that real-time PCR could detect addition of 106 cells to one
gram of faeces, provided that the target bacterium was not included in the original faecal
sample. Addition of 109 cells of Bifidobacterium longum to faeces resulted in no
difference compared with control sample, which had no added bacterial cells, due to the
pre-existence of B. longum in the original sample.
The two chemistries used in real-time PCR were equally sensitive; however,
successful usage of the intercalating dye SYBR Green I required applying of a hot start
polymerase to prevent formation of primer dimers, whereas with 5´-nuclease chemistry, a
similar sensitivity level was reached with a conventional DNA polymerase. Furthermore,
the 5´-nuclease chemistry was faster to perform because of its more straightforward
incubation protocol, one run taking approximately 80 min.
5.4. Suitability of L. brevis strains as dairy supplements (V)
Two Lactobacillus brevis strains, ATCC 8287 and ATCC 14869T, were evaluated
for their applicability as putative probiotics in dairy products. The strains tolerated well
low pH, bile acids and pancreatic fluid under in vitro conditions. They also expressed
good in vitro adherence to human Caco-2 and Intestine-407 cells. Adherence of the L.
brevis strains was especially strong to the small intestinal cell line, Intestine 407. In
antimicrobial activity assays, strain ATCC 8287 showed inhibitory properties towards
selected potentially harmful micro-organisms, particularly against Bacillus cereus.
Antimicrobial resistance tests revealed that both L. brevis strains were resistant to
vancomycin as well as to several other antibiotics, which is, however, typical for the
genus Lactobacillus. The L. brevis strains were unable to acidify milk to yoghurt but
were suitable as supplement strains in yoghurts. This was demonstrated by producing a
set of yoghurt products and analysing their rheological and sensory properties during a
cold storage period of 28 days. Despite its human origin, L. brevis ATCC 14869T did not
survive the gastrointestinal tract, whereas L. brevis ATCC 8287 was detected in faecal
samples taken during and immediately after ingestion of the strain. In conclusion, L.
brevis ATCC 8287 was considered a promising candidate for use as a probiotic adjunct in
dairy products.
35
6. Discussion
Molecular biology tools are needed for better understanding of the composition
and functions of the intestinal microbiota. In this study, different levels of detection were
developed for lactic acid bacteria and faecal microbiota.
Probiotic studies require careful documentation of the proposed effects. For better
monitoring of a potential strain, strain-specific discrimination could be beneficial in, for
example, feeding studies. Furthermore, specific recognition of industrial strains with
commercial value would help in monitoring the usage of these strains. Therefore,
Lactobacillus helveticus CNRZ32 was chosen as a model organism for demonstration of
genetic labelling. Although the strain in question does not fulfil the criteria set for a
probiotic, selection of this organism was considered justified for several reasons. The
strain is a starter strain used in the dairy industry, its proteolytic system has been studied
extensively (Dudley et al., 1996; Yüksel and Steele, 1996; Fenster et al., 1997; Shao et
al., 1997; Chen and Steele, 1998; Pederson et al., 1999) and methods for its
transformation and gene disruption have been developed (Bhowmik and Steele, 1993;
Chen and Steele, 1998). Various ‘food-grade’ markers for lactic acid bacteria have been
suggested, often with introduction of new phenotypic properties (Allison and
Klaenhammer, 1996; Geoffroy et al., 2000). However, creation of a label without
introduction of any phenotypic changes into the target strain would be more easily
accepted by consumers and legislators. Hertel et al. (1992) labelled a Lactococcus lactis
strain by introducing silent mutations into a plasmid-encoded gene. By contrast, to ensure
the stability of the created tag, we used a chromosomal gene for labelling of L. helveticus
CNRZ32. As demonstrated in Study I, a silent mutation site is effortlessly detected from
different matrices, such as faeces or milk, by PCR. Alternatively, hybridisation probes
could be utilised for detection (Hertel et al., 1992).
Direct detection of target nucleic acids present in population samples removes the
need for cultivation steps. Here, PCR-ELISA, dot blot hybridisation and real-time PCR
were compared for their suitability for analysis of population samples. Similar to PCRELISA method described in this paper, DGGE- and TGGE-based applications have been
found to detect approximately 1-10% subpopulations with PCR-amplified 16S rDNA
fragments when studying faecal samples (Zoetendal et al., 1998), suggesting their use for
36
analysis of predominant microbes of population samples. By using DGGE with groupspecific 16S rDNA primers, however, characterisation of diversity of smaller
subpopulations has been reached (Overmann et al., 1999; Walter et al., 2001; Heilig et
al., 2002). Similarly, the bifidobacteria-specific PCR-ELISA could successfully be used
for species-level analysis of faecal bifidobacteria; previous DGGE analysis results of the
same samples by Satokari et al. (2001b) generally support results obtained with PCRELISA. One drawback of PCR-ELISA identification has, however, been uncovered.
When results were compared with PCR-ELISA analysis, it soon became evident that
ado440 (Yamamoto et al., 1992), intended to be specific for B. adolescentis, also
recognized B. ruminantium observed in the faeces of some volunteers (Satokari et al.,
2001b). New sequence alignments confirmed that the ado440 probe had no mismatches
with B. ruminantium 16S rDNA. While the advantage of DGGE is the possibility for
further sequence analysis and identification of emerging new amplicons in DGGE
profiles, PCR-ELISA seems to be slightly more sensitive to changes in the relative
amounts of target species.
Strain-level studies have confirmed the complexity, uniqueness and stability of
bifidobacterial populations in the GI-tract (McCartney et al., 1996; Kimura et al., 1997;
Mangin et al., 1999). PCR-ELISA results were in accord with earlier reports. The
observed species distribution correlated well with previous European studies (Mangin et
al., 1994; Satokari et al., 2001a), and species distribution usually remained unchanged
within the samples of each subject. However, reduction of intensities of B. longum specific probes was observed frequently in the groups that ingested the probiotic B. lactis
Bb-12, although this could not be verified with statistical analysis, mainly due to the
small size of the test groups. This suggests that B. lactis Bb-12 may have partly replaced
B. longum during the ingestion period.
Hybridisation allows direct semi-quantitative monitoring of population samples
and thus avoids the problems caused by PCR bias. Dot blot hybridisation with rRNAtargeted oligonucleotide probes is a well-established method for studying faecal microbes
(Doré et al., 1998; Sghir et al., 2000; Hopkins et al., 2001; Marteau et al., 2001).
Ribosomal RNA is abundantly present in the bacterial cells, thus increasing the
sensitivity of the dot blot hybridisation to a reasonable level. However, radioactively
37
labelled probes are generally needed for successful detection of nucleic acid targets from
population samples. Here, dot blot hybridisation was performed with rDNA-targeted
oligonucleotide probes to quantify the same starting material as with the real-time PCR
applications tested. Dot blot hybridisation could, at its best, detect a 3% DNA
subpopulation from mixed DNA samples. A similar sensitivity level has been reported by
Muttray and Mohn (2000) for measurement of rDNA:rRNA ratios. Thus, the result was
as expected, being approximately 10-fold less sensitive than that obtained with rRNAtargeted oligonucleotides (Sghir et al., 2000). Real-time PCR was tested as an alternative
for a sensitive detection of target bacteria in faeces. The sensitivity level obtained for the
real-time PCR assays was 200-400 target bacteria in pure cultures or mixed DNA
samples, depending on the genome size of a target bacterium. Results obtained from
artificial DNA mixtures were confirmed by a reconstruction assay; addition of 106
bacterial cells to faecal samples containing approximately 1011 bacteria could be detected
with real-time PCR in a quantitative manner. The acquired level, providing a means for
quantifying a 0.01% subpopulation in a DNA sample, was considered sufficient for
studying the gastrointestinal tract ecology.
Although nucleic acid based detection methods offer a means to avoid bias caused
by cultivation or phenotypic testing of bacterial isolates, these techniques are not totally
devoid of problems. The main issues for detection of nucleic acids in mixed samples are
isolating DNA from different sources with constant efficiency, minimising the
occurrence of false-positive reactions due to contamination or poor planning of the
primers or hybridisation probes used, and avoiding negative reactions due to inhibiting
substances. Also, any oligonucleotides used as detection probes or primers should be
considered specific only in relation to the current sequence data. Several methods for
faecal sample treatment for isolation of DNA have been described (Biswas et al., 1994;
Klijn et al.; 1995; Shames et al., 1995; Wang et al., 1996; Lou et al., 1997; Monteiro et
al., 1997; Apajalahti et al., 1998). Nevertheless, this step remains somewhat problematic
because of the presence of PCR-inhibiting substances in the faeces and the overall
complexity of faecal microbial populations. Although over 90% recovery of the total
bacterial DNA present in faeces has been claimed (Apajalahti et al., 1998), non-selective
isolation of bacterial DNA from population samples is difficult to achieve or
38
demonstrate. Commercial kits have recently become available for isolation of faecal
DNA. However, McOrist et al. (2002) observed differences in the relative efficacy of
extraction of bacterial DNA from faeces with four commercial kits. Furthermore, the
relative sensitivities could not be extrapolated from DNA extractions performed directly
from pure cultures (McOrist et al., 2002).
Evaluation of possible probiotic properties of a bacterial strain requires intensive
and carefully planned testing of the strain both in vivo and in vitro. Although L. brevis is
not typically used as a probiotic, Kishi et al. (1996) have shown that oral administration
of live L. brevis ssp coagulans strain significantly stimulated the host immunity system
by increasing IFN-α production in human subjects. This result cannot, however, be
directly extrapolated to the L. brevis strains studied here. Adherence of lactic acid
bacteria to various components of human intestine has been studied with the human colon
adenocarcinoma Caco-2 cell line (Greene and Klaenhammer, 1994; Lehto and Salminen,
1997; Jacobsen et al., 1999; Kimoto et al., 1999), Intestine-407 cells derived from human
embryonic jejunum and ileum (Sarem et al., 1996), and intestinal mucus (Ouwehand et
al., 1999a, and b, 2001b). Both Caco-2 and Intestine-407 cells were used in the adhesion
assays for evaluation of the adhesion ability of L. brevis. These cell lines offer
complementary models for adhesion studies (Sarem et al., 1996). When compared with
the probiotic L. rhamnosus GG, adherence of the L. brevis strains was especially strong
to the small intestinal cell line, Intestine 407, suggesting that the small intestine could be
affected with the strains studied. L. brevis GRL62 has also been shown to have
intermediate adhesion properties to human intestinal mucus (Ouwehand et al., 2001b).
The two L. brevis strains studied were antagonistic towards some of the
potentially harmful micro-organisms, while they did not significantly inhibit the growth
of yoghurt-starter bacteria. In a previous study of Koga et al. (1998), L. brevis GRL1
(ATCC 8287), included in a panel of 41 Lactobacillus strains belonging to L. gasseri, L.
reuteri, L. casei, L. helveticus, L. brevis, L. fermentum and L. plantarum, weakly
inhibited two Vibrio cholerae strains of the eight strains tested. In the present study, L.
brevis GRL1 was shown to strongly inhibit B. cereus and to some extent also the growth
of S. aureus and other harmful micro-organisms chosen for the assay.
39
In vitro adhesion assays and testing for tolerance of low pH, bile acids and
pancreatic fluids have often been considered to be good indicators of the survival of a
bacterial strain through the GI tract. In this study, both L. brevis strains performed well in
the in vitro tests, and survival through the stomach could therefore be suggested for both
strains, especially when simultaneous ingestion of the bacteria with food, resulting in a
higher pH in the stomach, is taken into account. However, L. brevis GRL62 failed to
survive under in vivo conditions. This was surprising as the strain has originally been
isolated from human faeces (Orla-Jensen, 1919). In contrast, L. brevis GRL1, originally
isolated from fermented olives, could tolerate the gastrointestinal conditions and was
found to persist in the human gut.
Although the strains studied were not suitable for fermenting yoghurt,
supplementation of yoghurt with either of these strains had no negative effects on yoghurt
taste, appearance, or preservation. Therefore, L. brevis GRL1, which was able to survive
in the GI tract, could be considered for use as a supplementary strain in yoghurt or other
dairy products. To date, several commercial probiotics are already available and many
others are being studied, but no single strain is likely to possess all of the beneficial
properties suggested for probiotics. L. brevis GRL1, shown to be antagonistic towards
Bacillus cereus (this study) and Vibrio cholerae (Koga et al., 1998), could be a valuable
addition to the probiotics field. The surface (S)-layer of this strain is well-characterized
and potential applications for the S-layer have been suggested (Vidgrén et al., 1992;
Kahala et al., 1997; Savijoki et al., 1997; Kahala and Palva, 1999, Hynönen et al., 2002).
Recently, the S-layer has been shown to mediate the ability of the L. brevis GRL1 strain
to adhere to human intestinal, urinary bladder and endothelial cells (Hynönen et al.,
2002). Good adhesion properties could assist in the competitive exclusion of potentially
harmful microbes by L. brevis GRL1, and usage of this strain as an effective mucosal
vaccination tool because of its binding properties may be warranted. More studies are,
however, required to confirm these hypotheses.
40
7. Conclusions
Creation of a strain-specific nucleic acid tag by labelling of a chromosomal target
gene with a silent mutation and utilisation of the label for strain-specific detection of the
target strain was demonstrated using Lactobacillus helveticus CNRZ32. Although the
strain in question is not considered to be a probiotic, the method described here could be
used for probiotics. However, this requires the pre-existence or development of suitable
transformation tools as well as sequence data of a suitable target gene. From the
viewpoint of current legislation and consumer acceptance, the silent labelling method
should be the least unacceptable method for making genetic alterations to a bacterial
strain used in food processing, especially as the phenotypic properties of the labelled
strain were demonstrated to remain unchanged, thus attesting to the safety of the method.
PCR-ELISA with universal primers was shown to be suitable for detection of
predominant bacteria present in a mixed population sample. Specificity of the PCR step
proved to be critical for the sensitivity of the method; application of bifidobacterialspecific 16S primers enabled species- or group-specific detection of bifidobacteria
present in human faeces. The overall sensitivity of PCR-ELISA matched DGGE
approaches, with similar PCR primer specificities. Furthermore, being technically a
rather simple method, PCR-ELISA is easily transferred between different laboratories,
and could, in principle, be used in a similar manner as DGGE for analysis of large sample
numbers.
Real-time PCR was established to be a superior method for detection of single
bacterial species or larger groups from mixed DNA samples or faeces. Compared with
dot blot hybridisation with radioactive probes, real-time PCR provided an improved
sensitivity for detection, a means to avoid usage of radioactive labels, increased speed
and volume of sample analysis, an easier methodology and better reproducibility.
Detection of target bacteria added to faeces prior to the DNA isolation and purification
steps was demonstrated. In future, multiplexing with different labels and improvements
in hardware and software to allow simultaneous monitoring of a large number of PCR
reactions are likely to make real-time PCR an even more potent tool for population
analyses.
41
L. brevis GRL1 could be considered to be a potential probiotic strain. Although
several probiotics are already on the market, new strains with beneficial properties are
continually being sought. The strain in question has a well-characterized S-layer, and
offers promise as a potential mucosal vaccination strain.
42
8. Acknowledgements
This study was carried out at the Division of Microbiology, Department of Basic
Veterinary Science, Faculty of Veterinary Medicine, University of Helsinki, during
1998–2002. I wish to express my thanks to the numerous people who helped me during
this period. The following individuals deserve special mention:
My supervisor, Professor Airi Palva, whose guidance, support and ability to create
functional networks between research groups has been invaluable. Docent Ilkka Palva for
insightful comments and suggestions concerning planning of scientific tests, their
realisation, and finally, analysis of the outcome.
I have had the pleasure to share the laboratory with a wonderful group of people:
thank you all for your endurance and encouraging attitude. My former and present roommates, Reija Laitinen, Elina Rönkä, Johannes Aarnikunnas, Anna Kassinen and Teemu
Rinttilä, are especially thanked. I could never have survived without the help of our
research assistants and technical staff: Sinikka Ahonen’s contribution to my thesis, in
particular, has been of profound importance.
My thanks are due to the reviewers of this thesis manuscript, Professors Tapani
Alatossava and Finn Vogensen, whose comments improved this thesis immensely. I also
wish to express my sincere thanks to Maria Saarela and Jaana Mättö, members of my
follow-up group. In addition, the many researchers and industrial personnel with whom
I’ve had the pleasure to work are warmly acknowledged.
Carol Ann Pelli is thanked for editing the English language of this thesis.
Finally, I am grateful to my friends and family for ongoing support throughout
this process.
This work has been financed by the Technology Research Centre (TEKES) and
the Finnish Graduate School on Applied Bioscience: Bioengineering, Food & Nutrition,
Environment (ABS).
43
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